The small intestine epithelial cell is critical in regulating iron absorption, however, the molecular mechanisms of this process are poorly understood. The objective of this proposal is to characterize the normal mechanisms of heme and non-heme iron absorption and the mechanisms by which this process is regulated using an in vitro model. The model, the CRL 1592 small intestine cell strain, has features characteristic of small intestine epithelial cells and is a unique system for studying unidirection iron flow across the cell. The moieties which bind iron on the lumenal surface of the small intestine will be isolated and the molecular mechanisms involved in apical membrane transport characterized. The intracellular trafficking of iron will be examined by pulse-chase experiments followed by subcellular fractionation. The intracellular iron binding proteins and their interactions will be characterized by structural and chemical methods. Studies on the transfer of iron from its intracellular location into the extracellular "plasma" will utilize isolated basolateral membrane vesicles and, bicameral culture systems. Molecular biology techniques will be used in exploring gene expression as a function of iron stores for those proteins which have been correlated with iron absorption. The role of each of these proteins in partitioning iron trafficking will be examined after transforming small intestine cells with mini gene expression as a function of differentiation as cells mature along the villus crypt axis. Tissue specific gene expression will be examined by searching for alternative transcription initiation sites and transcription/translational gene control.